A Study on Trypanosomal Polyamine Biosynthetic Enzymes

Abstract: The polyamines, putrescine, spermidine, and spermine, are essential for all forms of life. The polyamine biosynthetic pathway has been shown to be a potential target for drugs against various trypanosomal parasitic diseases. The two main enzymes in the biosynthesis of polyamines are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). ODC catalyzes the first step of polyamine biosynthesis. Mammalian ODC is tightly regulated by polyamines and has a very fast turnover rate. The degradation of this enzyme is 26S proteasome dependent, but ubiquitin independent. The polyamines induce the degradation of mammalian ODC by stimulating the synthesis of a protein termed antizyme, which binds to and targets the enzyme for degradation by the 26S proteasome. Most trypanosomal ODCs, e.g. Leishmania donovani ODC and Trypanosoma brucei ODC are metabolically stable, but, interestingly, Crithidia fasciculata ODC has a fast turnover. In this thesis, the mechanisms involved in the rapid turnover of C. fasciculata ODC are examined. The degradation of C. fasciculata ODC was shown to be rapid also in mammalian systems, whereas that of L. donovani ODC was not. The rapid turnover of the enzyme was markedly reduced by inhibition of the 26S proteasome, indicating the involvement of this proteolytic system in the degradation of C. fasciculata ODC. However, unlike the mammalian ODC, C. fasciculata ODC was not down-regulated by polyamines, suggesting an antizyme independent degradation mechanism. As a result of analyzing the turnover of a series of chimeric ODCs between C. fasciculata ODC and L. donovani ODC, it was found that the central part of the former enzyme is important for its rapid degradation. The characterization of C. fasciculata AdoMetDC, the other main polyamine biosynthetic enzyme, revealed that also this enzyme has an extremely fast turnover (ca 3 min). Furthermore, no polyamine-mediated feedback regulation of AdoMetDC was observed in the parasite. As an alternative to using polyamine biosynthesis inhibitors, CHO cells expressing the trypanosomal ODCs were used to analyze the effects of different polyamine pools on cell cycle progression. It was shown that CHO cells expressing L. donovani ODC had a reduced polyamine synthesis, resulting in a decrease in cellular putrescine and spermidine levels (compared to wild-type CHO cells). On the other hand, the spermine content was increased. The changes in polyamine content were reflected in changes in cell growth kinetics.